Asymmetric divisions, in which a polarized cell divides to produce daughters with different fates, contribute to cell fate specification during development as well as stem cell maintenance. The proposed project addresses the molecular mechanisms of spindle positioning and cytokinesis during asymmetric divisions in the Caenorhabditis elegans embryo. As in other systems, asymmetric division in the C. elegans one-cell embryo relies on a conserved pathway in which the PAR polarity proteins regulate the distribution of components of a non-canonical G protein signaling pathway. We identified LET-99, a member of the DEPDC1 family, as a new player in this pathway. LET-99 is localized in an asymmetric cortical band pattern by the PAR-3 and PAR-1 proteins. LET-99 in turn restricts the cortical localization of the positive regulators of G protein signaling, GPR and LIN-5, to certain regions of the cell cortex. The asymmetric localization of these intermediates is an essential feature of spindle positioning, because GPR and LIN-5 associate with regulators of the microtubule motor dynein to generate the asymmetric cortical pulling forces that move the spindle. Once the spindle is positioned, it signals back to the cortex to determine the plane of cleavage. How the PAR proteins promote asymmetry of spindle positioning factors, and how the G protein pathway is integrated with other signaling mechanisms, remains to be elucidated. The experiments proposed in Aim 1 will define the molecular mechanisms by which the PAR proteins regulate LET-99 asymmetry. The hypothesis that PAR-1 directly phosphorylates LET-99 to inhibit its localization at the posterior cortex will be tested using in vitro kinase assays followed by in vivo transgenic studies. PAR-3 inhibits LET-99 localization at the anterior via a separate mechanism, which will be investigated using a combination of live-imaging and genetic analysis. LET-99 interacting proteins will also be tested for a role in LET-99 cortical anchoring. The goal of Aim 2 is to determine how the LET-99/G pathway is integrated with Rho-family GTPase signaling to properly position the cytokinesis furrow relative to the spindle. Quantitative analysis of localization patterns in mutants combined with biochemical interaction assays will be used to determine how these pathways interact. The hypothesis that LET-99 directly binds Rho GTPases via its partial RhoGAP domain will also be tested. Finally, Aim 3 will test the hypothesis that the human orthologs of LET-99, DEPDC1 and DEPDC1B, have a similar function to LET-99. Specifically we will test the hypothesis that these proteins associate with G or Rho and are involved in spindle movements or cytokinesis. Because of the conservation of pathway components, the results of these studies will be relevant to asymmetric division in many systems and will define the function of a novel class of proteins, the DEPDC1 family. PUBLIC HEALTH RELEVANCE: Asymmetric divisions and spindle positioning are critical for generating cell diversity during normal development. Recent studies also highlight the importance of asymmetric division in stem cell maintenance and cancer. All of the proteins under study are conserved. Further, the analysis of LET-99 in C. elegans shows that LET-99, a DEPDC1 family protein, is a novel regulator of G protein signaling. Therefore, the proposed studies of the mechanistic basis of spindle positioning in C. elegans and the biochemical function of LET-99 will greatly benefit health related-areas such as cancer and stem cell biology. In addition, DEPDC1 is upregulated in bladder cancer. The studies of DEPDC1 outlined in the proposal will begin to characterize the cell biological functions of this protein, which will help elucidate its role in cancer cells.